Chlorhexidine Inhibits L1 Cell Adhesion Molecule–Mediated Neurite Outgrowth in Vitro
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nature publishing group Articles Basic Science Investigation Chlorhexidine inhibits L1 cell adhesion molecule–mediated neurite outgrowth in vitro Aaron M. Milstone1, Penny Bamford2, Susan W. Aucott1, Ningfeng Tang2, Kimberly R. White2 and Cynthia F. Bearer2 BACKGROUND: Chlorhexidine is a skin disinfectant that Trace amounts of chlorhexidine can be absorbed through reduces skin and mucous membrane bacterial colonization the skin after a single bath in adults and term neonates. and inhibits organism growth. Despite numerous studies In 1976, Case (5) showed that, in adults, chlorhexidine “is assessing chlorhexidine safety in term infants, residual con- absorbed through intact human skin to an extraordinarily cerns have limited its use in hospitalized neonates, especially small degree, if at all.” This report only detected chlorhexidine low-birth-weight preterm infants. The aim of this study was in feces of adults following a hand wash with 5% chlorhexidine to assess the potential neurotoxicity of chlorhexidine on the digluconate. No chlorhexidine was detected in blood, indicat- developing central nervous system using a well-established ing minimal absorption. In term neonates, given a 1 or 2% in vitro model of neurite outgrowth that includes laminin and chlorhexidine bath, chlorhexidine was detected in 4 out of 20 L1 cell adhesion molecule (L1) as neurite outgrowth–promot- neonates (range: 13.5–26.7 ng/ml) (6). However, the interval ing substrates. between the bath and blood sampling was not reported, nor is METHODS: Cerebellar granule neurons are plated on poly it known when the chlorhexidine concentration may peak in L-lysine, L1, or laminin. Chlorhexidine, hexachlorophene, or the blood after topical exposure. There are no reports of imme- their excipients are added to the media. Neurons are grown for diate adverse consequences as a result of chlorhexidine absorp- 24 h, fixed, and neurite length is measured. tion in studies of term newborns or adults, no data to suggest RESULTS: Chlorhexidine significantly reduced the length that detectable serum concentrations have clinical importance of neurites grown on L1 but not on laminin. Chlorhexidine (7), and no data that chlorhexidine can cross the blood–brain concentrations as low as 125 ng/ml statistically significantly barrier. Another broad-spectrum organopolychlorinated anti- reduced neurite length on L1. Hexachlorophene did not affect septic, hexachlorophene (HEX), was found to penetrate intact neurite length. human skin and cause vacuolar encephalopathy in newborns CONCLUSION: Chlorhexidine at concentrations detected in in the 1970s (8,9), thus showing that antiseptics used with- the blood following topical applications in preterm infants spe- out harm in adults can cause devastating neurological injury cifically inhibited L1-mediated neurite outgrowth of cerebellar in infants. As a result, chlorhexidine has been scrutinized for granule neurons. It is now vital to determine whether the blood– its potential to be absorbed through the skin. Despite numer- brain barrier is permeable to chlorhexidine in preterm infants. ous studies assessing short-term outcomes from the use of chlorhexidine in term infants, residual concerns have limited hlorhexidine is a skin disinfectant that reduces skin and its use in hospitalized neonates, especially in low-birth-weight Cmucous membrane bacterial colonization and inhibits preterm infants (10). No developmental neurotoxicity stud- organism growth. It is frequently used to prevent infections ies have been performed to assess the safety of chlorhexidine and the spread of antibiotic-resistant bacteria, such as meth- exposure in term or preterm neonates. icillin-resistant Staphylococcus aureus (1,2). Chlorhexidine Our goal is to begin studies on the impact of chlorhexidine is used to prepare the skin before surgical procedures and exposure on the developing central nervous system. In the reduces the risk of surgical site infections (3). The Centers current study, our objective was to assess the potential neu- for Disease Control and Prevention recommend cleaning rotoxicity of chlorhexidine using a well-established in vitro the skin with chlorhexidine before the placement of central model of neurite outgrowth (11,12) that includes both lam- venous catheters, because numerous studies have demon- inin and L1 cell adhesion molecule (L1) as outgrowth-pro- strated reduced catheter infection rates following chlorhexi- moting substrates. L1-mediated, but not laminin-mediated, dine skin preparation as compared with alternatives such neurite outgrowth is dependent on lipid rafts, portions of the as povidone–iodine. However, “no recommendation can be plasma membrane similar to bacterial membranes disrupted made for the safety or efficacy of chlorhexidine in infants by chlorhexidine (13). Thus, L1-mediated neurite outgrowth aged <2 mo” (4). may be a sensitive indicator of chlorhexidine neurotoxicity. 1Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland; 2Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland. Correspondence: Cynthia F. Bearer ([email protected]) Received 17 January 2013; accepted 29 May 2013; advance online publication 13 November 2013. doi:10.1038/pr.2013.175 8 Pediatric RESEArch Volume 75 | Number 1 | january 2014 Copyright © 2014 International Pediatric Research Foundation, Inc. Chlorhexidine inhibits neurite outgrowth Articles HEX has been shown to cause axonopathy in vitro. Although 2). Ethanol was used as a positive control for inhibition of chemically distinct, both chlorhexidine and HEX contain the L1-mediated neurite outgrowth. We chose the highest concen- lipophilic moieties of polychlorinated phenol rings, mak- tration of chlorhexidine detected in the serum of a newborn ing them both lipophilic. Measurement of neurite outgrowth following topical exposure (1,021 ng/ml) (15). As can be seen may be one way to demonstrate the potential neurotoxicity of in Figures 1 and 2, compared with L1 control, CHG reduced chlorhexidine and HEX (14). neurite length of CGNs plated on L1 (P < 0.05). The vehicle (V) Due to concerns that chlorhexidine may cause neurotoxic- in which CHG is solubilized had no effect on neurite length. ity to third-trimester equivalent preterm infants, cerebellar There was no apparent effect on neurite branching under any granule neurons (CGNs) are an ideal in vitro model, as neurite of these conditions. outgrowth of these neurons occurs at this age of development. To determine whether these reagents reduce cell viability, we Neurite outgrowth is a sensitive measure for nonlethal neuro- counted the number of cells per high-powered field. Nonviable toxicity and provides an excellent model to assess the potential cells lose adhesion and lift off the plates. Thus, decreasing cellular toxicity of chlorhexidine (11,12). Neurite outgrowth number of adherent cells would indicate increased cell death. occurs through different mechanisms depending on environ- There was no difference in the number of cells adherent to the mental cues. These cues include proteins either on the surface plate as compared with poly l-Lysine (PLL), and no difference of other cells or in the extracellular matrix. L1 is one such envi- was found between the number of cells adherent to the plate ronmental cue. It binds homophilically to itself, initiating the in V alone or V with CHG (Figure 3). processes of protein trafficking, phosphorylation/dephosphor- HEX is a known developmental neurotoxicant (8,9,16). The ylation, and signal transduction in the growth cone. Laminin ability of HEX to inhibit L1-mediated neurite outgrowth was promotes neurite outgrowth by binding to the integrin recep- tested. The concentration of HEX used was 100× that in the tor and hence represents a different mechanism by which neu- serum of a newborn with liver disease (4,350 ng/ml) (17). rite outgrowth is promoted. Together, these cell lines provide We found no significant effect of either dimethyl sulfoxide an opportunity to investigate the potential neurotoxicity of (DMSO) alone or DMSO with 1 mmol/l HEX on L1-mediated chlorhexidine. neurite outgrowth (Figure 4). To determine whether the inhibition of neurite outgrowth RESULTS by CHG is specific to L1 or is a general effect on neurite out- We first tested whether chlorhexidine gluconate ((CHG) in a growth, we plated CGNs on laminin. Laminin is another cell proprietary vehicle (V)) inhibited L1-mediated neurite out- adhesion molecule that binds to integrin receptors. As can growth by plating CGNs on a substrate of L1 (Figures 1 and be seen in Figure 5, neither the vehicles nor CHG or HEX ab 20 µm 20 µm cd 20 µm 20 µm Figure 1. Chlorhexidine inhibits L1-mediated neurite outgrowth. Cerebellar granule neurons are plated on either poly L-lysine (PLL) alone or PLL plus L1-Fc (L1), and then, ethanol (E, final concentration: 25 mmol/l), chlorhexidine vehicle (V, final concentration: 1%), or chlorhexidine in vehicle (CHG, final concentration: 1.0 µg/ml CHG, 1% vehicle) are added to the media. The cells are grown for 24 h, fixed, and immunostained. Representative photomicro- graphs are shown: (a) PLL alone; (b) L1; (c) L1 + V; and (d) L1 + V + CHG. Original magnification: ×20. Copyright © 2014 International Pediatric Research Foundation, Inc. Volume 75 | Number 1 | january 2014 Pediatric RESEArch 9 Articles Milstone et al. 80 80 † 70 * 70 60 60 m) µ m) µ 50 50 40 40 † ite length ( ‡ ite length ( ** 30 30 Mean neur 20 Mean neur 20 10 10 0 PLL L1